Cargo crowding drives sorting stringency in COPII vesicles

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy dictates ER retention: in the absence of abundant cargo, non-specific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein, or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates lumenal steric pressure that drives selectivity. Sorting stringency is thus an emergent property of the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.